Cyclic mechanical stretch regulates the AMPK/Egr1 pathway in tenocytes via Ca2+-mediated mechanosensing

ABSTRACT

Purpose

Mechanical stimuli are essential for the maintenance of tendon tissue homeostasis. The study aims to elucidate the mechanobiological mechanisms underlying the maintenance of tenocyte homeostasis by cyclic mechanical stretch under high-glucose (HG) condition.

Materials and methods

Primary tenocytes were isolated from rat Achilles tendon and 2D-cultured under HG condition. The in vitro effects of a single bout, 2-h cyclic biaxial stretch session (1 Hz, 8%) on primary rat tenocytes were explored through Flexcell system. Cell viability, tenogenic gene expression, intracellular calcium concentration, focal adhesion kinase (FAK) expression, and signaling pathway activation were analyzed in tenocytes with or without mechanical stretch.

Results

Mechanical stretch increased tenocyte proliferation and upregulated early growth response protein 1 (Egr1) expression. An increase in intracellular calcium was observed after 30 min of stretching. Mechanical stretch phosphorylated FAK, calmodulin-dependent protein kinase kinase 2 (CaMKK2), and 5’ adenosine monophosphate-activated protein kinase (AMPK) in a time-dependent manner, and these effects were abrogated after blocking intracellular calcium. Inhibition of FAK, CaMKK2, and AMPK downregulated the expression of Egr1. In addition, mechanical stretch reinforced cytoskeletal organization via calcium (Ca2+)/FAK signaling.

Conclusions

Our study demonstrated that mechanical stretch-induced calcium influx activated CaMKK2/AMPK signaling and FAK-cytoskeleton reorganization, thereby promoting the expression of Egr1, which may help maintain tendon cell characteristics and homeostasis in the context of diabetic tendinopathy.

KEYWORDS:

• Tendon
• mechanotransduction
• intracellular calcium
• AMPK
• Egr1
• diabetic tendinopathy

Abbreviations

Table

Acknowledgments

We are grateful for the technical support provided by the staff of the P2 Lab of the Orthopedics Department, and the Microscopy Core Facility, Department of Medical Research, National Taiwan University Hospital.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Ethical approval

All the study procedures were approved by the Institutional Animal Care and Use Committee of the National Taiwan University College of Medicine and College of Public Health (IACUC Approval No. 20,160,402).

Author contributions

Yu-Ting Huang, Jui-Sheng Sun, and Yuan-Hung Chao conceptualized and designed this study. Yu-Ting Huang, Yu-Fu Wu, and Yi-Heng Chiu performed the experiments. Chung-Chen Jane Yao collected the resources. Hsing-Kuo Wang, Jui-Sheng Sun, and Yuan-Hung Chao contributed to data analysis and corrected the manuscript. All authors read and approved the final manuscript.

Additional information

Funding

This work was supported by research grant from the Ministry of Science and Technology, Taiwan (Grant number: MOST 106-2314-B-002-143), ROC.